用改进的重叠区扩增基因拼接法构建中间缺失突变体

Construction of middle fragment deletion mutant with improved gene splicing by overlap extension

目的 构建缺失第 32 5～ 373位氨基酸密码子的磷脂酸磷酸化激酶β(phosphatidylinositol 4 - kinase beta,PI4 K-β)基因 ,以探索一种简便的构建中间缺失突变体的方法。 方法 应用重叠区扩增基因拼接法的原理 ,在拼接延伸后 ,再进行一次 PCR扩增 ,得到缺失第 32 5～ 373位氨基酸密码子的 PI4 K- β基因 ,克隆至 p CMV- Tag4 A真核表达载体。 结果 成功地构建出缺失第 32 5～ 373位氨基酸密码子的 PI4 K- β突变体。 结论 这种改进的重叠区扩增基因拼接法可以有效而可靠地构建中间缺失突变体 ,具有推广价值。

ObjectiveTo construct a phosphatidylinositol 4-kinase beta (PI4K-β) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant. MethodsIn line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-β mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.ResultsA mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.ConclusionThe improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.